Hydrogen peroxide stimulates a tyrosine kinase-dependent calcium release from intracellular stores, which is assumed to be achieved through the activation of phospholipase Cgamma2 (PLCgamma2) via a tyrosine phosphorylation mechanism in B cells. Here we show that hydrogen peroxide induces both tyrosine phosphorylation on PLCgamma2 and the activation of phosphatidylinositol 3-kinase (PI3K) in B cells and that the PI3K inhibitor, Wortmannin, partially inhibited the hydrogen peroxide-induced calcium release without affecting tyrosine phosphorylation on PLCgamma2. Overexpression of human Bruton's tyrosine kinase (Btk), which was activated by hydrogen peroxide, almost completely overcame the inhibition of calcium release by Wortmannin. The reversal of Wortmannin's inhibition by enhancing Btk concentration seemed unique to the hydrogen peroxide-mediated effect because Btk failed to overcome the inhibition of Wortmannin on B cell receptor-triggered calcium mobilization. Immunoblot analysis revealed that Btk formed stable complexes with several tyrosine-phosphorylated proteins, including PLCgamma2, only in Btk overexpressed cells upon hydrogen peroxide stimulation. Together, our data are consistent with the notion that PIP3 and/or a high concentration of Btk targets the activated PLCgamma2 to its substrate site for maximal catalytic efficiency.That Btk overexpression overcomes the inhibitory effect of Wortmannin on hydrogen peroxide-induced calcium mobilization implicates a possible role of Btk in the regulation of calcium signaling. We have Btk-deficient DT40 cells and established the stable cell lines expressing wild-type Btk or Btk mutants either in kinase (Arg525 to Gln), Src homology 2 (SH2, Arg307 to Ala), or pleckstrin homology (PH, Arg28 to Cys) domains. Using these mutants, we are investigating the roles and structure-function relationship of Btk in hydrogen peroxide-induced calcium mobilization.